This invention relates to a process for determining the presence and/or concentration of a material in a small sample by utilizing at least two fluorescent indicators in the sample and detecting fluorescent color changes of at least one indicator in the sample.
Detection or measurement of chemical parameters such as pH and of chemical reactant concentrations by optical methods is well established. Such methods are generally valuable in chemistry, biology, medicine and related fields, for purposes of detection, measurement and related diagnostic tests. Optical measurement or detection is recognized as particularly valuable because optical methods are generally non-contacting, often nonperturbing, reasonably specific and very rapid. More specifically, optical methods such as colorimetry or light absorbance are particularly valuable for use with diagnostic tests in medicine and other fields, because a quantitative measurement or a simple, rapid, visual inspection allows determination or detection of a diagnostic result.
The process of colorimetric optical measurement consists of measuring the absorption of light by a sample at different wavelengths. Colorimetric optical indicators have the desirable property of being easily measured, or of exhibiting a readily distinguishable color change by simple visual inspection. Typically, a sample is illuminated by optical radiation containing a wide range of wavelengths, such that relatively different amounts of absorption are revealed as a readily perceived color. A general property of colorimetric detection or measurement is that the intensity of light transmitted by a sample is governed approximately by Beer's Law, which is given below. EQU I(.lambda.)=I.sub.o C.sup..epsilon.(.lambda.)Cd
where I(.lambda.) is the transmitted light intensity, I.sub.o (.lambda.) is the incident light intensity, .epsilon.(.lambda.) is the molar extinction coefficient, C is the concentration of the optical indicator or dye, d is the optical path length and .lambda. is the light wavelength. An important consequence is that colorimetric detection or measurement of small amounts or concentrations of chemical indicators, or of chemical reactant, is essentially obtained from the difference or ratio of two large parameters, the first parameter being the incident optical intensity and the second parameter being the transmitted optical intensity. It is well known that detection or measurement based on a small difference, or on ratios, between two large parameters is generally inaccurate, because the magnitude of the noise or error in each of the two large parameters tends to be large compared to the difference or ratio in the two large parameters. In essence colorimetric measurement involves measurement in the presence of a large background intensity, whereas fluorescence measurement does not. For these reasons, a significant disadvantage of colorimetric optical measurement or detection is that the sensitivity or detection limit for the underlying chemical indicator or reactant concentration is much poorer than for fluorescence measurement.
A further, related disadvantage applies to colorimetric detection or measurement carried out on very small size samples. This is important, because it is becoming generally desirable to carry out chemical detection and measurement using small samples, particularly in biological and medical tests. As indicated by Beer's Law, the logarithm of the ratio, y [I(.lambda.)/I.sub.o (.lambda.)], of transmitted to incident optical intensity is proportional to the product of three quantities: (1) .epsilon.(.lambda.), the molar extinction coefficient, which is a property of a particular chemical indicator species or of a particular chemical reactant, (2) C, the concentration of the indicator or reactant, and (3) d, the optical path length within the sample. In the important case of small samples, most particularly samples with a maximum linear dimension of about 1000 micron (10.sup.-1 cm) down to about 1 micron (10.sup.-4 cm), the indicator or reactant concentration must be significantly larger than is the case for conventional measurement using conventional optical cuvetts with a standard optical path length of 1.0 cm. The desirability of detection and measurement in small samples such as liquid or gel microdroplets is increasingly recognized as disclosed, for example, in U.S. Pat. Nos. 4,399,219 and 4,401,755 and in Enzyme Engineering 7, Vol. 434, reprinted from Annals of the New York Academy of Sciences, pp. 363-372, Weaver et al. Small samples which are approximately spherical in shape have maximum optical path lengths equal to the spherical diameter, so that for the case of optical path lengths in the range 10.sup.-1 to 10.sup.-4 cm, the corresponding sample volumes are very small, in the range 5.2.times.10.sup.-4 ml to 5.2.times.10.sup.-13 ml. More specifically, for small samples with optical path lengths in this range of 10.sup.-1 to 10.sup.-4 cm, in order to obtain the same perceived color, or to make a measurement with essentially the same optical accuracy, the indicator or reactant concentration must be present at a concentration which is a factor about 10.sup.` to 10.sup.4 larger than in the conventional arrangement using a 1 cm path length. This results in the undesirable degredations of the detection or measurement ability by the same factor of about 10.sup.1 to 10.sup.4 for colorimetric detection or measurement in small samples.
An important related problem arises when chemical indicator species are employed to detect or measure parameters such as pH. The requirement of a large concentration of indicator species because of the small optical path length often results in a degredation of detection or measurement performance because of an interaction of the highly concentrated indicator with a primary chemical reaction within the small volume. For example, in the important case of detection or measurement of acid production from a single or small number of micororganisms or other biochemically active entity within a small volume sample, the presence of a high concentration of pH indicator species can significantly increase the buffering capacity within the small sample, and thereby significantly reduce the ability to rapidly detect or measure acid production with the small volume sample.
In the important case of direct, visual inspection, colorimetric measurement based on color change has the desirable property of allowing simple, rapid assessment of whether or not a chemical change has occured. For example, in the well-known and important case of acid-base determination wherein pH is used as an indicator, there are a variety of well-known colorimetric pH indicators which are known to give useful perceived color changes over a useful range of pH values. Examples of colorimetric pH indicators and typical corresponding ranges of useful pH are methyl violet (pH 0.1-1.5), bromphenol blue (pH 3.0-4.6), methyl red (pH 4.8-6.0), bromothymol blue (pH 6.0-7.6), phenolphthalein (pH 8.2-10) and 1,3,5-trinitobenzene (pH 11.5-15.0). In contrast, visual inspection as the basis of the change in absolute intensity of transmitted light at one relatively narrow band of wavelengths is significantly more difficult, because it is more difficult to visually determine changes in optical intensity than to determine changes in color.
Fluorescence measurement is often based on changes in intensity of the emitted light for fixed excitation light conditions. Relatively few fluorescent indicators exhibit a large change in the relative strength of emission as a function of wavelength as a chemical parameter such as pH varies. Instead, fluorescence measurements are generally carried out on the basis of changes of total intensity, using only a single fluorescent species as a chemical indicator or a chemical reactant. In contrast to the availability of colorimetric indicators, fluorescent species which respond to changes in an exemplary chemical parameter such as pH generally usually exhibit only a large variation in strength of total fluorescence emission, which property, therefore, does not allow a determination to be made on the basis of a perceived color change.
For example, Kirkbright, "Fluorescent Indicators" in Indicators, R. Belcher and H. Frieser (Eds.), Pergamo Press, Oxford, pp. 685-708, (1972), lists seventy six (76) fluorescent indicators for pH, of which only twenty-four (24) actually change emission color, while the remaining thirty-two (32) only change emission intensity at the same color. Further, of these twenty-four (24), only eight (8) are potentially useful in the broad physiological pH range of about 5&lt;pH&lt;9, and only four (4) are potentially useful in the more important physiological range 6&lt;pH&lt;8. Finally, the pH range over which the color change of a single fluorescent indicator species is significant is generally broad, making visual observation difficult (Kirkbright, 1972), and the pH at which the maximum change occurs may not correspond to a desirable pH, and cannot be significantly altered. Several other fluorescent pH indicators, which generally have these properties are described by Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Junction City, (1985).
For example, in the case of intracellular pH measurements using fluorescence, wherein the cell volume comprises a small volume sample, it has been found to be generally necessary to use complex instrumentation to exploit relatively small change in the relative emission in different wavelength bands of the emission spectrum of a single dye which is taken up or contained within a living cell. However, the small changes exploited in these intracellular pH measurements are generally too small to be used as the basis of a readily perceived color change. Instead, a relatively complex measurement procedure must be used. In partial summary, the advantage of a readily perceived color change is generally not obtained from the established use of fluorescence indicators or fluorescent products of chemical reactions.
The overall teaching of the prior art is that in the case of measurement of reactant concentrations by fluorescence means in small volume samples, a single fluorescent species is used if a reactant is measured or determined. Similarly, the overall teaching of the prior art is that in the case of measurement based on fluorescent chemical parameters in small volume samples, a single fluorescent species is used as an indicator.
Accordingly, it would be highly desirable to provide a simple-to-use optical indicator methodology which would have significantly better sensitivity or detection limits than conventional colorimetry for chemical parameter or chemical reactant concentration while still retaining the advantage of a color change while still providing an indicator means which can also be advantageously employed in instruments and to provide improved fluorescence means of measuring indicator or reactant concentrations in small volume samples.